Crystal structure of the integral membrane diacylglycerol kinase

Abstract

Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

Figure 1. Crystal structure of Δ4 DgkA

a, Structure (ribbon model) viewed from the membrane plane. Individual subunits are coloured blue, green and orange. Membrane boundaries are based on hydrophobic thickness calculations from the PPM server. b. As in a, viewed from the cytoplasm. Putative active sites are designated based on the identities of the subunits (A, B, C) contributing to each site and demarked with a seven pointed star. c. As in b with helices shown as cylinders and the structure notation introduced. The solid black triangle marks the axis of an approximate three-fold symmetry. Dotted triangles define the transmembrane helical core of the trimer (black) and the arrangement of transmembrane helices (H1-H3) in individual subunits (white). The N-terminus (N) in the model of each subunit is identified. It leads directly into an amphiphilic surface helix (SH) whose length varies between subunits.

Figure 2. Rationalizing functional biochemistry with the crystal structure of DgkA

Residues in DgkA where mutations to cysteine reduce kinase activity to 7 % or less that of wild type activity are mapped onto the crystal structure of Δ4 DgkA. a, View in the membrane plane. The protein (ribbon model) is displayed with chains A, B and C colored blue, green and orange, respectively. Residues are shown in ball and stick and color coded according to atom type (nitrogen, blue; oxygen, red; carbon, grey). b, View from the cytoplasm. c, An expanded view of the putative active site (asAB) of the enzyme, where residues critical to kinase activity are clustered.

Figure 3. Putative active site of DgkA complete with lipid and Mg²⁺-ATP substrates and activating zinc

a, A putative active site of Δ4 DgkA is shown superposed with 7.8 MAG lipid substrate, the zinc ion from Δ7 DgkA, and Mg²⁺-ATP from the docking calculation. The ATP (thick sticks, atoms colored by type) extends ∼20 Å from the cytoplasmic loop between H2 and H3 to the polar headgroup of the lipid substrate (large spheres, atoms colored by type) at the polar/apolar interface of the membrane. Residues in the vicinity of the substrates that are known to be critical for kinase activity are shown in ball and stick, as in Fig. 2. Zinc and magnesium are shown as brown and blue spheres, respectively. b, Expanded view of a with the ribbon model removed for clarity.

Figure 4. Comparison of the crystal and solution NMR structures of DgkA

A view of the crystal (left) and solution structures (right) from a, the cytoplasm, and b, the membrane plane. c, An expanded view of the putative active site region. The five most highly conserved residues and how they contribute to a single active site (asBC) in the solution structure are shown (right panels), as described by Van Horn et al.. In the crystal structure, three of these five residues (Glu 69, Asn 72, Glu 76) contribute to one active site, asBC; two (Lys 94, Asp 95) are in asAB. The SH of the solution model, which is ill-defined, has been truncated for reasons of space. SH_B of the crystal structure has been trimmed at residue 18 to reveal the active site (asBC). Membrane boundaries are based on hydrophobic thickness calculations from the PPM server.